Fc mutants with modified functional activity

ABSTRACT

Disclosed is a polypeptide including a mutated Fc region and having functional activity, mediated by the Fc region, that is modified compared with that of a parent polypeptide. The Fc region includes at least one combination of 2 mutations, the combination being selected from among one mutation selected from among a first set of mutations, and at least one mutation selected from among a second set of mutations, and provided that mutation (i) does not take place on the same amino acid as mutation (ii). Also disclosed are use of the polypeptide, compositions including the same, and methods for preparing the polypeptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 15/554,022,filed on Aug. 28, 2017, which is the National Phase under 35 U.S.C. §371 of International Application No. PCT/FR2016/051068, filed on May 6,2016, which claims the benefit under 35 U.S.C. § 119(a) to PatentApplication No. 15 54101, filed in France on May 7, 2015, all of whichare hereby expressly incorporated by reference into the presentapplication.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (7111-0439 SequenceListing.xml; Size: 18,264 bytes; and Date of Creation: Mar. 29, 2023) isherein incorporated by reference in its entirety.

The present invention relates to a polypeptide comprising a mutated Fcregion and having functional activity, mediated by the Fc region, thatis modified compared with that of a parent polypeptide.

An antibody is formed of a tetramer of heavy and light chains. The twolight chains are identical, and the two heavy chains are identical andlinked by disulfide bridges. There are five types of heavy chains(alpha, gamma, delta, epsilon, mu) which determine immunoglobulinclasses (IgA, IgG, IgD, IgE, IgM). The light chain group comprises twosub-types: lambda and kappa.

IgGs are soluble antibodies which can be found in the blood and otherbody fluids. IgG is a Y-shaped glycoprotein with an approximatemolecular weight of 150 kDa, composed of two heavy chains and two lightchains. Each chain is characterized by a constant region and a variableregion. The two carboxy-terminal domains of the heavy chains form the Fcfragment, whilst the amino-terminal domains of the heavy and lightchains recognize the antigen and are known as the Fab fragment.

Fc fusion proteins are created by a combination of an antibody Fcfragment with a protein domain which provides specificity for a giventherapeutic target. Examples are combinations of the Fc fragment withall types of therapeutic proteins or fragments thereof.

Therapeutic antibodies and Fc fusion proteins are currently used totreat various diseases such as rheumatoid polyarthritis, psoriasis,multiple sclerosis and numerous forms of cancer. Therapeutic antibodiesmay be monoclonal or polyclonal antibodies. Monoclonal antibodies areobtained from a unique antibody cell production line which exhibitsidentical specificity for a single antigen. Therapeutic Fc fusionproteins are used or developed as medicinal products against autoimmunediseases, e.g. etanercept (Enbrel by Amgen, a TNF receptor linked to anFc fragment) or Alefacept (Amevive by Biogen Idec: LFA-3 linked to theFc portion of human IgG1).

So that antibodies and Fc fusion proteins are able to exert theireffects, the Fc fragment must have the best activity possible (e.g.antibody-dependent cell cytotoxicity, complement-dependent cytotoxicityor antibody-dependent cell phagocytosis) i.e. it is optimised. Saidoptimisation would allow a protein to be obtained having improvedactivity and/or efficacy and/or reduced side effects.

The Applicant has henceforth developed particular Fc fragments havingimproved activity. These fragments can be used for therapy to impartimproved efficacy to a product containing the same.

The present invention therefore provides a variant of a parentpolypeptide, having optimised properties related to the functionalactivity mediated by the Fc region.

The present invention relates to a polypeptide comprising a mutated Fcregion and having functional activity, mediated by the Fc region, thatis modified in comparison with that of a parent polypeptide, said Fcregion comprising at least one mutation selected from among: G316D,K326E, N315D, N361H, P396L, T350A, V284L, V323I, P352S, A378V, Y436H,V266M, N421T, G385R, K326T, H435R, K447N, N434K, K334N, V397M, E283G,A378T, F423L, A431V, F423S, N325S, P343S, K290E, S375R, F405V, K322E,K340E, N389S, F243I, T307P, N389T, S442F, K248E, Y349H, N286I, T359A,S383R, K334R, T394P, V259A, T393A, P352L, Q418P, V302A, L398P, F423P,S442P, V363I, S383N, S254F, K320E, G402D, 1253F, V284A, A431T, N315H,Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R,V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G,V262A, V305A, K246R, V308I, E380G, N276S, K439Q, S267G, F423Y, A231T,K320R, L410R, K320M, V412M, T307N, T366A, P230S, Y349S, A339T, K246E,K274E, A231P, 1336T, S298N, L234P, S267N, V263A, E333G, V308A, K439R,K392R, S440G, V397I, 1336V, Y373D, K288E, L309P, P227S, V379A, K288R,K320T, V282A, 1377T, N421S and C261R, the numbering being that of the EUindex or Kabat equivalent.

Said polypeptide is called «polypeptide of the invention» in the presentapplication.

More specifically, the present invention relates to a polypeptidecomprising a mutated Fc region and having functional activity, mediatedby the Fc region, that is modified in comparison with that of a parentpolypeptide, said Fc region comprising at least one combination of 2mutations, said combination being selected from among:

-   -   (i) one mutation selected from among 307N, 326E, 326T, 334N,        334R, 352L, 378V, 378T, 394P, 396L, 397M and 421T;    -   (ii) at least one mutation selected from among 226Y, 227S, 230S,        231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F,        255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R,        276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R,        290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G,        315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G,        334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S,        359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T,        379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P,        396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T,        421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,        435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,        the numbering being that of the EU index or Kabat equivalent,        provided that mutation (i) does not take place on the same amino        acid as mutation (ii).

Preferably, mutation (i) is selected from among 378V, 396L and 397M.Preferably, the polypeptide also comprises a mutation selected fromamong 248E, 326T, 333G and 423Y.

Preferably, mutation (ii) of the invention is selected from among 226Y,227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F,255W, 259A, 261R, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A,283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P,308A, 308I, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I,325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G,383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 398P, 405V,405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V,431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N.

Throughout this present application, the numbering of the residues inthe Fc region is the numbering of the immunoglobin heavy chain inaccordance with the EU Index or equivalent in Kabat et al. (Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991). The expression «EUIndex or Kabat equivalent» refers to the EU numbering of the residues ofhuman IgG1, IgG2, IgG3 or IgG4 antibodies. This is can be found on theIMGT website(http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html).

By «polypeptide» or «protein» is meant a sequence comprising at least100 covalently bonded amino acids.

By «amino acid», is meant one of the 20 natural amino acids ornon-natural analogues.

The term «position» means a position in the sequence of a polypeptide.For the Fc region, the positions are numbered according to the EU indexor Kabat equivalent.

The term «antibody» is used in its common meaning. It corresponds to atetramer comprising at least one Fc region and two variable regions.Antibodies particularly comprise full-length immunoglobulins, monoclonalantibodies, multi-specific antibodies, chimeric antibodies, humanisedantibodies and entirely human antibodies. The amino-terminal portion ofeach heavy chain comprises a variable region of about 100 to 110 aminoacids and responsible for recognition of the antigen. In each variableregion, three loops join together to form a binding site to the antigen.Each of the loops is called a complementarity determining region (CDR).The carboxy-terminal portion of each heavy chain defines a constantregion chiefly responsible for the effector function.

IgGs have several sub-classes, in particular IgG1, IgG2, IgG3 and IgG4.The IgM sub-classes are particularly IgM1 and IgM2. Therefore, by«isotype» is meant one of the immunoglobulin sub-classes defined by thechemical and antigenic characteristics of their constant regions. Theknown isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4,IgA1, IgA2, IgM1, IgM2, IgD and IgE.

Full-length IgGs are tetramers and are composed of two identical pairsof two immunoglobulin chains, each pair having a light chain and a heavychain, each light chain comprising the VL and CL domains, and each heavychain comprising the domains VH, Cγ1 (also called CH1), Cy2 (also calledCH2) and Cy3 (also called CH3). For a human IgG1, «CH1» refers topositions 118 to 215, «CH2» refers to positions 231 to 340 and «CH3»refers to positions 341 to 447 in the EU index or Kabat equivalent. Theheavy chain of IgGs also comprises an N-terminal flexible hinge regionwhich refers to positions 216 to 230 for IgG1. The lower hinge regionrefers to positions 226 to 230 in the EU Index or Kabat equivalent.

By “variable region” is meant the region of an immunoglobulin whichcomprises one or Ig domains substantially encoded by any of the genesVK, Vλ and/or VH which make up the kappa, lambda and heavyimmunoglobulin chains, respectively. The variable regions comprise thecomplementarity determining regions (CDRs) and framework regions (FRs).

The term “Fc” or “Fc region” designates the constant region of anantibody with the exclusion of the first immunoglobulin constant regiondomain (CH1). Therefore, Fc refers to the two last domains (CH2 and CH3)of the IgGI constant region, and to the N-terminal flexible hinge ofthese domains. For a human IgG1, the Fc region corresponds to residueC226 as far as its carboxy-terminal end, i.e. the residues at positions226 to 447, numbered as in the EU index or Kabat equivalent. The Fcregion used may further comprise part of the upper hinge region locatedbetween positions 216 to 226 in the EU Index or Kabat equivalent; inthis case, the Fc region used corresponds to the residues at positions216 to 447, 217 to 447, 218 to 447, 219 to 447, 220 to 447, 221 to 447,222 to 447, 223 to 447, 224 to 447 or 225 to 447, numbered as in the EUIndex or Kabat equivalent. Preferably, in this case, the Fc region usedcorresponds to the residues at positions 216 to 447, as numbered in theEU Index or Kabat equivalent.

Preferably, the Fc region used is selected from among the sequences SEQID NO: 1 to 10.

By «parent polypeptide» is meant a reference polypeptide. Said parentpolypeptide may be of natural or synthetic origin. Within the context ofthe present invention, the parent polypeptide comprises an Fc regioncalled «parent Fc region D. This Fc region can be selected from amongthe group of wild-type group Fc regions, the fragments or mutantsthereof. Preferably, the parent polypeptide comprises a human Fc region,preferably an Fc region of a human IgG1. The parent polypeptide maycomprise pre-existing modifications of amino acids in the Fc region(e.g. an Fc mutant) compared with wild-type Fc regions.

Advantageously, the parent polypeptide parent is an isolated Fc region(i.e. an Fc fragment as such), a sequence derived from an isolated Fcregion, an antibody, a fusion protein comprising an Fc region, or an Fcconjugate, this list being nonlimiting. By «sequence derived from anisolated Fc region» is meant a sequence comprising at least two isolatedFc regions that are linked together such as an scFc (single chain Fc) ormultimer Fc. By «fusion protein comprising an Fc region» is meant apolypeptide sequence fused to an Fc region, said polypeptide sequencepreferably being selected from among the variable regions of anyantibody, the binding sequences of a receptor to its ligand, adhesionmolecules, ligands, enzymes, cytokines and chemokines. By «Fc conjugate»is meant a compound that is the result of the chemical coupling of an Fcregion with a conjugation partner. The conjugation partner may beproteinic or non-proteinic. The coupling reaction generally usesfunctional groups on the Fc region and the conjugation partner. Variousbinding groups are known in the prior art as being appropriate forsynthesis of a conjugate: for example, homo- or heterobifunctionalbinding groups are well known (see, catalogue of the Pierce ChemicalCompany, 2005-2006, technical section on cross-linking agents, pages321-350). Among suitable conjugation partners, mention can be made oftherapeutic proteins, labels, cytotoxic agents such as chemotherapeuticagents, toxins and the active fragments thereof. Suitable toxins andtheir fragments notably include the diphtheria toxin, exotoxin A, ricin,abrin, saporin, gelonin, calicheamicin, auristatins E and F, andmertansine. Advantageously, the parent polypeptide—and hence thepolypeptide of the invention—consists of an Fc region.

Advantageously, the parent polypeptide—and hence the polypeptide of theinvention—is an antibody.

Finally, preferably, the parent polypeptide—and hence the polypeptide ofthe invention—is a polypeptide produced in the milk of transgenicanimals.

By «mutation» is meant a change of at least one amino acid in thesequence of a polypeptide, in particular a change of at least one aminoacid in the Fc region of the parent polypeptide. The mutated polypeptideobtained is a variant polypeptide; it is a polypeptide of the invention.Said polypeptide comprises a mutated Fc region in comparison with theparent polypeptide. Preferably, the mutation is a substitution,insertion or deletion of at least one amino acid. By «substitution» ismeant the replacement of one amino acid at a particular position in aparent polypeptide sequence by another amino acid. For example,substitution N434S refers to a variant polypeptide, here a variant inwhich asparagine at position 434 is replaced by serine. By “insertion ofamino acid” or “insertion” is meant the addition of an amino acid at aparticular position in a parent polypeptide sequence. For example,insertion G>235-236 designates an insertion of glycine between positions235 and 236. By «deletion of amino acids» or «deletion» is meant thedeletion of an amino acid at a particular position of a parentpolypeptide sequence. For example, E294del designates the deletion ofglutamic acid at position 294. Preferably, the following designation isused for mutation: «434S» or «N434S», and indicates that the parentpolypeptide comprises asparagine at position 434 replaced by serine inthe variant. If there are a combination of substitutions, the preferredformat is the following: «259I/315D/434Y» or «V259I/N315D/N434Y». Thisindicates that there are three substitutions in the variant, atpositions 259, 315 and 434, and that the amino acid at position 259 ofthe parent polypeptide i.e. valine is replaced by isoleucine, that theamino acid at position 315 of the parent polypeptide i.e. asparagine isreplaced by aspartic acid and that the amino acid at position 434 of theparent polypeptide i.e. asparagine is replaced by tyrosine.

The polypeptide of the invention has functional activity, mediated bythe Fc region, that is modified in comparison with that of the parentpolypeptide.

By “functional activity mediated by the Fc region” is particularly meantthe effector functions. The functional activity mediated by the Fcregion therefore particularly comprises Antibody-Dependent Cell-mediatedCytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC),Antibody-Dependent Cell Phagocytosis (ADCP), endocytosis activity,cytokine secretion, or a combination of at least two of theseactivities. Preferably, the functional activity mediated by the Fcregion under consideration in the invention is selected from among ADCC,CDC and the combination thereof. This functional activity can beevaluated using methods well known in the prior art such as thosedescribed in the examples (see in particular items 4.4 and 4.5 of theexamples).

The functional activity mediated by the Fc region of the polypeptide ofthe invention is increased or reduced compared with that of the parentpolypeptide.

According to a first variant, the polypeptide of the invention hasfunctional activity mediated by the Fc region that is increased incomparison to that of the parent polypeptide. Preferably, thepolypeptide of the invention has functional activity mediated by the Fcregion that is increased in relation to the parent polypeptide by aratio of at least 2, preferably higher than 5, preferably higher than10, preferably higher than 15, preferably higher than 20, preferablyhigher than 25 and preferably higher than 30.

According to a second variant, the polypeptide of the invention hasfunctional activity mediated by the Fc region that is reduced comparedwith that of the parent polypeptide. Preferably, the polypeptide of theinvention has functional activity mediated by the Fc region that isreduced in relation to that of the parent polypeptide by a ratio of atleast 2, preferably higher than 5, preferably higher than 10, preferablyhigher than 15, preferably higher than 20, preferably higher than 25 andpreferably higher than 30.

Preferably, the mutated Fc region of the polypeptide of the inventionhas modified affinity for at least one of the receptors (FcRs) of the Fcregion, selected from among the C1q complement, FcgRIIIa (CD16a),FcgRIIa (CD32a) and FcgRIIb (CD32b). The C1q complement is involved inCDC activity. The FcgRIIIa receptor (CD16a) is involved in ADCC; itexhibits V/F polymorphism at position 158. The FcgRIIa receptor (CD32a)is involved in platelet activation and phagocytosis; it exhibits H/Rpolymorphism at position 131. Finally, the FcgRIIb receptor (CD32b) isinvolved in inhibition of cell activity. Preferably, said mutated Fcregion has increased affinity for at least one of the FcRs. Preferably,the affinity is increased compared with that of the parent Fc by a ratioof at least 2, preferably higher than 5, preferably higher than 10,preferably higher than 15, preferably higher than 20, preferably higherthan 25 and preferably higher than 30. In other words, the affinity ofthe mutated Fc region for an FcR is higher than that of the parentpolypeptide. Alternatively, said mutated Fc region has reduced affinityfor at least one of the FcRs. Preferably affinity is reduced in relationto that of the parent Fc by a ratio of at least 2, preferably higherthan 5, preferably higher than 10, preferably higher than 15, preferablyhigher than 20, preferably higher than 25 and preferably higher than 30.In other words, the affinity of the mutated Fc region for an FcR islower than that of the parent polypeptide. The FcR affinity of apolypeptide comprising an Fc region can be evaluated with methods wellknown in the prior art. For example, persons skilled in the art candetermine affinity (Kd) using surface plasma resonance (SPR).Alternatively, skilled persons can perform a suitable ELISA assay.Suitable ELISA assay allows a comparison between the binding forces ofthe parent Fc and mutated Fc. The detected signals specific to themutated Fc and parent Fc are compared. Binding affinity can bedetermined indifferently whether evaluating whole polypeptides orisolated Fc regions thereof.

Preferably, the mutated Fc region of the invention comprises 1 to 20mutations compared with the parent polypeptide, preferably 2 to 20mutations. By «1 to 20 modifications of amino acids» this encompasses 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20mutations of amino acids. Preferably, it comprises 1 to 15 mutations,preferably 2 to 15 mutations, preferably 1 to 10 mutations in relationto the parent polypeptide, preferably 2 to 10 mutations.

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises at least one combination of 2 mutations, said combinationbeing selected from among:

-   -   (i) one mutation selected from among 378V, 396L and 397M;    -   (ii) at least one mutation selected from among 231V, 248E, 286I,        286Y, 290E, 298N, 308A, 315D, 316D, 326E, 333G, 334N, 334R,        336T, 352S, 361H, 366A, 378T, 396L, 397M, 412M, 421T, 423Y and        447N,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises at least one combination of 2 mutations, said combinationbeing selected from among:

-   -   (i) one mutation selected from among 378V, 396L et 397M;    -   (ii) at least one mutation selected from among 248E, 316D, 326E,        333G, 378T, 396L and 421T,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the polypeptide of the invention comprises a mutated Fcregion having increased ADCC and CDC functional activities compared withthose of the parent polypeptide, characterized in that said Fc regioncomprises at least one combination of 2 mutations, said combinationbeing selected from among:

-   -   (i) one mutation selected from among 378V, 396L and 397M;    -   (ii) at least one mutation selected from among 248E, 316D, 326E,        333G, 378T, 396L and 421T,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises at least one combination of 2 mutations, said combinationbeing selected from among:

-   -   (i) mutation 378V;    -   (ii) at least one mutation selected from among 298N and 336T,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises at least one combination of 2 mutations, said combinationbeing selected from among:

-   -   (i) one mutation selected from among 378V, 396L and 397M;    -   (ii) at least one mutation elected from among 231V, 286I, 286Y,        290E, 315D, 334N, 352S, 361H, 366A, 378T, 397M, 412M, 421T and        423Y,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the polypeptide of the invention comprises a mutated Fcregion having increased ADCC functional activity compared with that ofthe parent polypeptide, characterized in that said Fc region comprisesat least one combination of 2 mutations, said combination being selectedfrom among:

-   -   (i) one mutation selected from among 378V, 396L and 397M;    -   (ii) at least one mutation selected from among 231V, 286I, 286Y,        298N, 290E, 315D, 334N, 336T, 352S, 361H, 366A, 378T, 397M,        412M, 421T and 423Y,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises at least one combination of 2 mutations, said combinationbeing selected from among:

-   -   (i) mutation 378V;    -   (ii) at least one mutation selected from among 248E, 308A, 334R,        447N, the numbering being that of the EU Index or Kabat        equivalent, and provided that mutation (i) does not take place        on the same amino acid as mutation (ii).

Preferably, the polypeptide of the invention comprises a mutated Fcregion having increased CDC functional activity compared with that ofthe parent polypeptide, characterized in that said Fc region comprisesat least one combination of 2 mutations, said combination being selectedfrom among:

-   -   (i) mutation 378V;    -   (ii) at least one mutation selected from among 248E, 308A, 334R,        447N,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises a combination of mutations selected from among thecombinations:

-   -   K320E/T394P/G402D    -   K290E/K320E/T350A/P396L    -   T359A/S383R/V397M

Preferably, the mutated Fc region of the polypeptide of the inventionhas improved affinity for the C1q complement, and comprises at least onecombination of 2 mutations, said combination comprising:

-   -   i) one mutation selected from among 378V, 378T, 396L, 421T, 334R        and 326E; and    -   ii) at least one mutation selected from among 361H, 290E, 316D,        248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T,        396L, 286I, 315D and 397M,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventionhas improved affinity for the FcgRIIIa receptor (CD16a), and comprisesat least one combination of 2 mutations, said combination comprising:

-   -   i) one mutation selected from among 378V, 326E, 397M, 334N and        396L; and    -   ii) at least one mutation selected from among 316D, 397M, 334N,        248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P,        307P, 378V, 378T, 286I, 286Y and 298N,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventionhas increased affinity for the FcgRIIa receptor (CD32a), and comprisesat least one combination of 2 mutations, said combination comprising:

-   -   i) one mutation selected from among 378V, 326E, 397M, 307N,        394P, 326T, 396L and 334N; and    -   ii) at least one mutation selected from among: 316D, 334R, 334N,        323I, 231V, 246R, 336T, 378T, 286Y, 286I, 352S, 383R, 359A,        421T, 361H, 315D, 366A, 290E, 307P and 439R,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, mutation (ii) is selected from among: 316D, 334R, 334N,323I, 231V, 246R, 336T, 378T, 286Y, 286I, 352S, 383R, 359A, 421T, 361H,366A, 290E, 307P and 439R.

Preferably, the mutated Fc region of the polypeptide of the inventionhas increased affinity for the FcgRIIb receptor (CD32b), and comprisesat least one combination of 2 mutations, said combination comprising:

-   -   i) one mutation selected from among 326E, 326T, 378V, 397M,        352L, 394P, 396L and 421T; and    -   ii) at least one mutation selected from among 316D, 334R, 248E,        334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventionhas increased CDC activity, and comprises at least one combination of 2mutations, said combination comprising:

-   -   i) one mutation selected from among 378V, 378T, 396L, 421T, 334R        and 326E; and    -   ii) at least one mutation selected from among 361H, 290E, 316D,        248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T,        396L, 286I, 315D and 397M,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventionhas increased ADCC activity, and comprises at least one combination of 2mutations, said combination comprising:

-   -   i) one mutation selected from among 378V, 326E, 397M, 334N and        396L; and    -   ii) at least one mutation selected from among 316D, 397M, 334N,        248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P,        307P, 378V, 378T, 286I, 286Y and 298N,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the mutated Fc region of the polypeptide of the inventioncomprises at least one combination of 3 mutations, said combinationcomprising:

-   -   (i) one mutation selected from among 326E, 326T, 352L, 378V,        378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and    -   (ii) at least 2 mutations selected from among 226Y, 227S, 230S,        231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F,        255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R,        276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R,        290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G,        315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G,        334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S,        359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T,        379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P,        396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T,        421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,        435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, the at least 2 mutations (ii) are selected from among 226Y,227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F,255W, 259A, 261R, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A,283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P,308A, 308I, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I,325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G,383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 398P, 405V,405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V,431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P, and 447N.

More preferably, the mutated Fc region of the polypeptide of theinvention comprises at least one combination of 4 mutations, saidcombination comprising at least one mutation (i) such as describedabove, and at least 3 mutations (ii) such as described above, thenumbering being that of the EU Index or Kabat equivalent, and providedthat mutation (i) does not take place on the same amino acid as mutation(ii).

More preferably, the mutated Fc region of the polypeptide of theinvention comprises at least one combination of 5 mutations, saidcombination comprising at least one mutation (i) such as describedabove, and at least 4 mutations (ii) such as described above, thenumbering being that of the EU Index or Kabat equivalent, and providedthat mutation (i) does not take place on the same amino acid as mutation(ii).

A further subject of the present invention is a composition ofpolypeptides of the invention, said purified polypeptides on theirAsn297 glycosylation site having N-glycans with a fucosylation ratelower than 65%, preferably lower than 50%, more preferably lower than40%. Preferably said purified polypeptides, on their Asn297glycosylation site, have a glycan structure of biantennary type withshort chains, low sialylation, having non-intercalary terminal mannosesand/or non-intercalary terminal N-acetylglucosamines.

More preferably, said purified polypeptides have a content higher than60% for the G0+G1+G0F+G1F forms, the content being lower than 50% forthe G0F+G1F forms. More preferably, said purified polypeptides have acontent higher than 60% for the G0+G1+G0F+G1F forms, the fucose contentbeing lower than 65%,

More preferably, said purified polypeptides have a content lower than40% for the G1F+G0F forms.

A further subject of the present invention is a pharmaceuticalcomposition comprising (i) a polypeptide of the invention or acomposition such as described in the preceding paragraph and (ii) atleast one pharmaceutically acceptable excipient.

A further subject of the present invention is the polypeptide of theinvention or the composition such as previously described for use asmedicinal product.

As previously indicated, advantageously the parent polypeptide—and hencethe polypeptide of the invention—is an antibody. In this case, theantibody can be directed against a tumour antigen, viral antigen,bacterial antigen, fungal antigen, a toxin, membrane-bound orcirculating cytokine, and a membrane receptor.

When the antibody is directed against a tumour antigen, it isparticularly suitable for use in the treatment of cancers. By «cancer»is meant any physiological condition characterized by abnormal cellproliferation. Examples of cancers notably include carcinoma, lymphoma,blastoma, sarcoma (including liposarcoma), neuroendocrine tumours,mesothelioma, meningioma, adenocarcinoma, melanoma, leukaemia andmalignant lymphoid pathologies, this list being non-exhaustive.

When the antibody is directed against a viral antigen, it isparticularly suitable for use in the treatment of viral infections.Viral infections in particular are infections due to HIV, a retrovirus,Coxsackie virus, smallpox virus, influenza virus, yellow fever virus,West Nile virus, to a cytomegalovirus, to a rotavirus or to thehepatitis B or C virus, this list being non-exhaustive.

When the antibody is directed against a toxin, it is particularlysuitable for use in the treatment of bacterial infections e.g.infections with the tetanus toxin, diphtheria toxin, Bacillus anthracistoxins, or for the treatment of infections with botulin toxins, ricintoxins, shiga toxins, this list being non-exhaustive.

When the antibody is directed against a cytokine, it is particularlysuitable for use in the treatment of inflammatory and/or autoimmunediseases. Inflammatory and/or autoimmune diseases notably includethrombotic thrombocytopenic purpura (TTP), graft or transplantrejection, graft-versus-host-disease, rheumatoid polyarthritis, systemiclupus erythematosus (SLE), different types of sclerosis, primarySjögren's syndrome (or Gougerot-Sjögren syndrome), autoimmunepolyneuropathies such as multiple sclerosis, type I diabetes, autoimmunehepatitis, ankylosing spondylitis, Reiter's syndrome, gouty arthritis,celiac disease, Crohn's disease, chronic Hashimoto thyroiditis(hypothyroidism), Addison's disease, autoimmune hepatitis, Basedow'sdisease (hyperthyroidism), ulcerative colitis, vasculitis such asANCA-associated systemic vasculitis (AntiNeutrophil CytoplasmicAutoantibodies), autoimmune cytopenia and other haematologicalcomplications in adults and children such as autoimmune acute or chronicthrombopenia, autoimmune haemolytic anaemia, haemolytic disease of thenewborn (HDN), cold agglutinin disease, autoimmune acquired haemophilia;Goodpasture syndrome, membranous nephropathy, autoimmune skin blisteringdisorders, refractory myasthenia, mixed cryoglobulinemia, psoriasis,juvenile chronic arthritis, inflammatory myositis, dermatomyositis andsystemic autoimmune disorders in children including paediatricantiphospholipid syndrome, connective tissue disease, autoimmune lunginflammation, Guillain-Barre syndrome, chronic inflammatorydemyelinating polyneuropathy (CIDP), autoimmune thyroiditis, mellitis,myasthenia gravis, autoimmune ocular inflammatory disease, neuromyelitisoptica (Devic's disease), scleroderma, pemphigus, diabetes throughinsulin resistance, polymyositis, Biermer's anaemia, glomerulonephritis,Wegener's disease, Horton disease, polyarteritis nodosa (PAN) andChurg-Strauss syndrome, Still's disease, atrophic polychondritis,Behcet's disease, monoclonal gammopathy, Wegener granulomatosis, lupus,haemorrhagic rectocolitis, psoriatic arthritis, sarcoidosis collagenouscolitis, dermatitis herpetiformis, familial Mediterranean fever, laglomerulonephritis with IgA deposits, Lambert-Eaton myasthenic syndrome,sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome anduveo-meningo-encephalitic syndrome.

Other inflammatory diseases are also included, such as for example theacute respiratory distress syndrome (ARDS), acute septic arthritis,adjuvant arthritis, allergic encephalomyelitis, allergic rhinitis,allergic vasculitis, allergy, asthma, atherosclerosis, chronicinflammation due to chronic bacterial or viral infections, chronicobstructive pulmonary disease (COPD), coronary disease, encephalitis,intestinal inflammatory diseases, inflammatory osteolysis, inflammationassociated with acute and delayed hypersensitivity reactions,inflammation associated with tumours, peripheral nerve lesion ordemyelinising diseases, inflammation associated with tissue trauma suchas burns and ischaemia, inflammation due to meningitis, multiple organdysfunction syndrome (MODS), pulmonary fibrosis, septicaemia and septicshock, Stevens-Johnson syndrome, undifferentiated arthritis, andundifferentiated spondyloarthropathies.

In one particular embodiment of the invention, the autoimmune disease isidiopathic thrombocytopenic purpura (ITP) and chronic inflammatorydemyelinating polyneuropathy (CIDP).

A further subject of the invention is a method to produce a polypeptidecomprising an Fc region and having functional activity, mediated by theFc region, that is modified in comparison with that of a parentpolypeptide, said method comprising a step to introduce at least onemutation in said Fc region, selected from among: G316D, K326E, N315D,N361H, P396L, T350A, V284L, V323I, P352S, A378V, Y436H, V266M, N421T,G385R, K326T, H435R, K447N, N434K, K334N, V397M, E283G, A378T, F423L,A431V, F423S, N325S, P343S, K290E, S375R, F405V, K322E, K340E, N389S,F243I, T307P, N389T, S442F, K248E, Y349H, N286I, T359A, S383R, K334R,T394P, V259A, T393A, P352L, Q418P, V302A, L398P, F423P, S442P, V363I,S383N, S254F, K320E, G402D, 1253F, V284A, A431T, N315H, Y319H, C226Y,F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R,M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A,K246R, V308I, E380G, N276S, K439Q, S267G, F423Y, A231T, K320R, L410R,K320M, V412M, T307N, T366A, P230S, Y349S, A339T, K246E, K274E, A231P,1336T, S298N, L234P, S267N, V263A, E333G, V308A, K439R, K392R, S440G,V397I, 1336V, Y373D, K288E, L309P, P227S, V379A, K288R, K320T, V282A,1377T, N421S and C261R, the numbering being that of the EU Index orKabat equivalent.

Preferably, the subject of the present invention is a method to producea polypeptide comprising an Fc region and having functional activity,mediated by the Fc region, that is modified in comparison with that of aparent polypeptide, said method comprising a step to introduce at leastone combination of 2 mutations, said combination being selected fromamong:

-   -   (i) one mutation selected from among 326E, 326T, 352L, 378V,        378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and    -   (ii) at least one mutation selected from among 226Y, 227S, 230S,        231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F,        255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R,        276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R,        290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G,        315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G,        334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S,        359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T,        379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P,        396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T,        421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,        435R, 436H, 439R, 440G, 440N, 442F, 442P, and 447N,    -   the numbering being that of the EU Index or Kabat equivalent,        provided that mutation (i) does not take place on the same amino        acid as mutation (ii).

Preferably, mutation (ii) is selected from among 226Y, 227S, 230S, 231V,234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R,262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I,286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G,309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N,334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H,362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S,389T, 392R, 393A, 393I, 394P, 396L, 397I, 398P, 405V, 405L, 410R, 412M,414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,435R, 436H, 439R, 440G, 440N, 442F, 442P, and 447N.

A further subject of the present invention is a method to increase thebinding of a polypeptide comprising an Fc region with at least one ofthe receptors (FcR) of the Fc region selected from among the receptorsC1q, FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b), said methodcomprising a step to introduce at least one mutation in said Fc region,selected from among:

G316D, K326E, N315D, N361H, P396L, T350A, V284L, V323I, P352S, A378V,Y436H, V266M, N421T, G385R, K326T, H435R, K447N, N434K, K334N, V397M,E283G, A378T, F423L, A431V, F423S, N325S, P343S, K290E, S375R, F405V,K322E, K340E, N389S, F243I, T307P, N389T, S442F, K248E, Y349H, N286I,T359A, S383R, K334R, T394P, V259A, T393A, P352L, Q418P, V302A, L398P,F423P, S442P, V363I, S383N, S254F, K320E, G402D, 1253F, V284A, A431T,N315H, Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V,K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H,D312G, V262A, V305A, K246R, V308I, E380G, N276S, K439Q, S267G, F423Y,A231T, K320R, L410R, K320M, V412M, T307N, T366A, P230S, Y349S, A339T,K246E, K274E, A231P, 1336T, S298N, L234P, S267N, V263A, E333G, V308A,K439R, K392R, S440G, V397I, 1336V, Y373D, K288E, L309P, P227S, V379A,K288R, K320T, V282A, 1377T, N421S and C261R, the numbering being that ofthe EU Index or Kabat equivalent.

Preferably, the subject of the present invention is a method to increasethe binding of a polypeptide comprising an Fc region to at least one ofthe receptors (FcR) of the Fc region, selected from among the receptorsC1q, FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b), said methodcomprising a step to introduce at least one combination of 2 mutations,said combination being selected from among:

-   -   (i) one mutation selected from among 326E, 326T, 352L, 378V,        378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and    -   (ii) at least one mutation selected from among 226Y, 227S, 230S,        231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F,        255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R,        276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R,        290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G,        315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G,        334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S,        359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T,        379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P,        396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T,        421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,        435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,        the numbering being that of the EU Index or Kabat equivalent,        and provided that mutation (i) does not take place on the same        amino acid as mutation (ii).

Preferably, mutation (ii) is selected from among 226Y, 227S, 230S, 231V,234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R,262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I,286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G,309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N,334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H,362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S,389T, 392R, 393A, 393I, 394P, 396L, 397I, 398P, 405V, 405L, 410R, 412M,414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N.

The sequences described in the present application can be summarised asfollows:

SEQ ID NO: Protein 1 Fc region of human IgGI, G1m1, 7 (residues 226-447in EU Index or Kabat equivalent) without N-terminal upper hinge region.2 Fc region of human IgG2 without N-terminal upper hinge region. 3 Fcregion of human IgG3 without N-terminal upper hinge region. 4 Fc regionof human IgG4 without N-terminal upper hinge region. 5 Fc region ofhuman IgG1, G1m3, without N-terminal upper hinge region. 6 Fc region ofhuman IgG1, G1m1,17, with N-terminal upper hinge region (residues216-447 in EU Index or Kabat equivalent). 7 Fc region of human IgG2 withN-terminal upper hinge region. 8 Fc region of human IgG3 with N-terminalupper hinge region. 9 Fc region of human IgG4 with N-terminal upperhinge region. 10 Fc region of human IgG1, G1m3, with N-terminal upperhinge region. 11 and 12 Primers MG_619 and MG_621

The present invention will be better understood on reading the followingexamples.

EXAMPLES Example 1: Identification of Polypeptides with Mutated FcRegion of the Invention and Characterization of Said Polypeptides

I. Material and Methods

1. Construction of Banks of the Human Fc Region

The human Fc gene encoding the amino acids 226-447 (EU Index or Kabatequivalent) i.e. a polypeptide comprising an Fc region, derived from aheavy chain of human IgG1 and having the allotype G1 m1.17 (SEQ ID NO:1), (Poul M A et al, Eur J. Immunol 25 (7): 2005-2009, 1995) was clonedin the phagemid vector pMG58 (pMG58_Fc226) as BamHI/EcoRI fragmentfollowing standard PCR protocols. Several fully randomized banks weregenerated applying the MUTAGEN™ method (WO02/038756) which useslow-fidelity human DNA-polymerases (mutases) to introduce randommutations homogeneously in the entire target sequence. Three differentmutases (pol R, pol n and pol i) were used under different conditions tocreate complementary mutation profiles. These human polymerases wereproduced and purified as previously described (Mondon et al. J.Biotechnol 21: 76-82 (2007), Emond et al. Protein Eng Des Sel21:267-274, (2008)).

1.1. Mutagenesis with the MUTAGEN™ Method

The MUTAGEN™ method was described in application WO02/038756.

In brief, the human Fc gene (Fc gene) was replicated with mutases usingthe 5′ primer MG_619: 5 ′-AGTACTGACTCTACCTAGGATCCTGCCCACCGTGC-3′ (SEQ IDNO: 11) and the 3′ primer MG_621: 5′-ACTGCTCGATGTCCGTACTATGCGGCCGCGAATTC-3′ (SEQ ID NO: 12). A mixturecontaining 0.6 μg of the pMG58_Fc226 plasmid as model (wild-type Fcregion or selected variants), the primers MG_619 and MG_621 (250 nMeach) and suitable replication buffer (see details below) was treatedfor 5 min at 95° C. and immediately cooled to 4° C. to denature the DNAstrands. For pol R, the replication buffer was 50 mM Tris HCl pH 8.8, 10mM MgCl2, 10 mM KCl, 1 mM DTT and 1% (v/v) glycerol. The replicationbuffer for pol η (or pol η and pol ι) was 25 mM Tris HCl pH 7.2, 5 mMMgCl2, 10 mM KCl, 1 mM DTT and 2.5% (v/v) glycerol. After the denaturingstep, mutagenic replications were obtained by adding 50 μM ATP/dCTP, 100μM dTTP/dGTP and 1 μg of pol β or 100 μM of dNTP and 1 μg of pol η (orpol η and pol ι, 1 μg of each mutase). The replication reaction wasconducted at 37° C. for two hours. The replication products weresubsequently concentrated and desalted on Microcon columns (Millipore).

1.2. Selective Amplification and Cloning of the Mutated Fragments

The previously obtained replication products were amplified by selectivePCR with tail primers. The primers (MG_619 MG_621) were designed with atail that was non-complementary to the model allowing specificamplification of the DNA fragments synthesized by the mutases. Onefraction of the replication products was added to a mixture containingthe PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 10 pmolof the 5′ and 3′ primers, 200 μM of dNTPs and 1.25 U of Platinum Taq DNApolymerase (Invitrogen). The PCR cycles were: first cycle 2 min. at 94°C., 10 sec. at 64° C., 30 sec. at 75° C., 1 min. at 94° C., followed by30 selective cycles: 20 sec. at 94° C. and 30 sec. at 75° C.

The amplified replication products were purified on 1% (w/v) of agarosegels digested with BamHI and EcoRI and cloned in the pMG58 vector. Theligation mixtures were transformed in E. Coli XL1-Blue electrocompetentcells and spread over a solid 2YT medium (16 g/l peptone, 10 g/l yeastextract, 5 g/l NaCl, 15 g/l agar) to which were added 100 μg/ml ofampicillin and 1% (w/v) glucose. After growth, the number of colonieswas determined to estimate the size of the banks and at least 48 clonesper bank were subjected to PCR and fast DNA sequencing. The cells werere-suspended in the 2YT medium with 15% glycerol, frozen and stored at−80° C.

1.3. Construction of the Mut3 Bank

A first bank was obtained using pol β on the wild-type Fc gene andcontained 3.2×10⁶ clones (called Mut1.1). The DNA of this first bank wasused to generate the second and third banks, respectively using pol β(3.8×10⁶ clones, Mut1.2) and pol η and i (3.0×10⁶ clones, Mut1.3).

This strategy with two accumulated replication steps allowed an increasein the mutation rate. The fourth bank was obtained with pol η alone onthe wild-type Fc gene (1.0×10⁶ clones, Mut1.4). Finally, these fourbanks were proportionately mixed to obtain the final bank called Mut1,representing 1.1×10⁷ different clones.

Two different banks were then constructed using a DNA pool of single anddouble mutants isolated on the FcRn receptor. A first bank was obtainedusing pol β (1.9×10⁷ clones, Mut2.1) and a second bank with pol η (1×10⁶clones, Mut2.2). Finally, these two banks were proportionately mixed toobtain the final bank called Mut2, i.e. 2×10⁷ different clones.

A new bank was obtained using pol β on the wild-type Fc gene, with adiversity of 4.7×10⁷ clones.

Finally, the banks Mut1, Mut2 and the new bank were proportionatelymixed to obtain the final bank called Mut3, i.e. 7.8×10⁷ differentclones.

The Mut3 bank finally contained 3×10⁷ mutated clones in phase with amean of 2.2 mutated amino acids per Fc.

1.4. Construction of the Mut4sel Bank

A bank of Fc variants was constructed from 36 clones having improvedbinding with C1q and/or CD16aV, selected from the Mut3 bank Mut3 (1.6mutated amino acids per Fc on average). The Mut4 bank was obtained usingan equimolar mixture of pol β and pol polymerases (in the replicationbuffer of pol β). The Mut4 bank has a diversity of 1.3×10⁷ clones, with77%, of clones in phase and on average 2.6 mutated amino acids per Fc.

This bank was cloned in the pMG93 vector, a selection vector developedfor Fc which allows selection of the coding sequences (ORFs) on anampicillin medium through fusion with the gene of □-lactamase. Thisconstruction was transformed in the non-suppressive bacteria HB2151,which allowed removal of the TAGs codons that are known to be stopcodons in this strain. The clones of this bank were spread plated in lowdensity in dishes of agar medium containing small amounts of ampicillin,to carry out selection of the ORFs, the viable clones then beingrecovered and frozen. Spread plating in 500 Petri dishes (120×120)allowed covering of the entire bank Mut4. The bank thus selected wasdenoted Mut4sel and contained 92%, clones in phase, i.e. 1×10⁷ mutatedclones in phase, with an average of 2.5 mutated amino acids per Fc. TheMut4sel bank was then sub-cloned in the phagemid pMG58, in XL1-Bluebacteria, to allow selection by «phage display» on the targets.

1.5. Construction of the Mut5 Bank

A bank of Fc variants was constructed from 42 clones having improvedbinding with C1q and/or one of the FcgR receptors, selected from theMut4sel bank (2.4 mutated amino acids per Fc on average). The Mut5 bankwas obtained using the polymerase pol β alone so as to obtain a lowmutation rate. Finally, the Mut5 bank obtained contained 1.2×10⁷ cloneswith 95% clones in phase and on average 3.1 mutated amino acids per Fc.The good quality of this bank did not require the selection of ORFs aswas required for the Mut4 bank.

2. Phage Display Expression of the Fc Banks and Selection of theImproved Variants

At the selection steps, the banks Mut3, Mut4sel and Mut5 were expressedon the surface of the M13 bacteriophage following standard procedures(Smith G P, Science 228: 1315 (1985)). E. coli XL1-Blue bacteria,containing the bank to be expressed cloned in the pMG58 vector, werecultured in 60 ml of 2YT medium to which were added 100 μg/mlampicillin, 15 μg/ml tetracycline and 1% (w/v) glucose at 30° C. Thecells were then infected with the auxiliary phage M13 (M13K07, Biolabs,bacteria/phage ratio=1:3) at 37° C. for 20 min and the production ofFc-phage was continued overnight at 26° C., at 230 rpm2YT/ampicillin/glucose with 0.5 mM IPTG and 50 μg kanamycin/ml. Thefollowing day, the phages were precipitated with PEG6000 followingstandard procedures, re-suspended in 1 ml of PBS buffer at pH 7.4 andtitrated by infecting XL1-Blue cells.

2.1 Recombinant Proteins Used:

The C1q complement is commercially available (Calbiochem).

CD16a is an activator receptor having V/F polymorphism at position 158,on the binding side to Fc. Affinity is improved for CD16aV.

CD16aV is commercially available (R&D system).

CD16aF was produced by PX′Therapeutics.

CD32a is an activator receptor having H/R polymorphism at position 131,on the binding site to Fc. Affinity is improved for CD32aH.

CD32aR is commercially available (R&D system).

CD32aH was produced by PX′Therapeutics. CD32b is an inhibitor receptorhaving lesser affinity for IgG1s than CD32aR. It is commerciallyavailable (R&D system).

2.2. Solid Phase Selections:

For solid phase selections, the Fc-phages diluted in PBS/5% skimmilk/0.1% Tween 20 were incubated in 8 wells of Maxisorp plates(1-4×10¹¹ phages/well in final 100 μl) previously coated with 500ng/well of CD16aV, biotinylated CD16aV, biotinylated CD16aF,biotinylated C1q, biotinylated CD32aR or biotinylated CD32b and blockedwith 5% skim milk in PBS. After incubation for 2 hours at 37° C., thewells were washed 8 times with PBS/0.1% Tween 20, and 2 times with PBS.For selections on CD32aR and CD32b, counter-selection rounds were alsocarried out: before the binding step on the target immobilised on aMaxisorp plate, the Fc-phages were pre-incubated in similar manner on 8wells with the competitive receptor. Only the non-bonded Fc-phages onthe first target were then transferred into wells containing the secondtarget as previously described. The selected phages were eluted byinfection with XL1-Blue bacteria in exponential growth phase (2×150μl/well, 20 min. at 37° C. without agitation). The infected bacteriawere spread plated on a solid 2YT/ampicillin/glucose medium. Thefollowing day, the cells were re-suspended in 2YT medium with 15%glycerol, frozen and stored at −80° C. until the following selectionround.

2.3. Liquid Phase Selections:

For liquid phase selections, 4×10¹¹ phages were first incubated withbiotinylated CD16aV (250 nM), biotinylated CD16aF (1000 nM), or withbiotinylated C1q (250 nM) for 1 hour at ambient temperature under lightagitation. Magnetic beads coated with streptavidin (Dynal) previouslyblocked with 5% skim milk in PBS were then added to the phages for 30minutes at ambient temperature. The phage-bead complexes were washed 10times with PBS/0.1 Tween 20 using a magnet. The phage-bead complexeswere used to infect 5 ml of XL1-Blue bacteria in exponential growth thatwere spread plated on a solid 2YT/ampicillin/glucose medium. Thefollowing day, the cells were re-suspended in 2YT medium with 15%glycerol, frozen and stored at −80° C. until the following selectionround.

2.4. Selections from Banks:

Mut3 Bank:

For liquid phase selections, 6 rounds were performed on 3 biotinylatedtargets: CD16aV, CD16aF and C1q (clones denoted DL6A, DL6B and QL6A).For the solid phase selection rounds, 6 rounds were performed on 3targets: CD16aV, biotinylated CD16aV and biotinylated C1q (clonesdenoted DS6A, DS6B and QS6A).

Mut4sel Bank:

The selection rounds were solely conducted in solid phase: 3 selectionrounds on biotinylated CD16aV, 3 rounds on biotinylated C1q, 3 rounds onbiotinylated CD32aR (+/−depletion on CD32b for the 3^(rd) round) and 3rounds on biotinylated CD32b (+/−depletion on CD32aR for the 3^(rd)round), (clones denoted A3A, G3A, J3A/B and K3A/B).

Mut5 Bank:

The selection rounds were conducted as previously: 3 selection rounds onbiotinylated CD16aF, 3 rounds on biotinylated C1q, 3 rounds onbiotinylated CD32aR, 3 rounds on biotinylated CD32b, 2 rounds onbiotinylated CD32aR and 2 rounds with depletion on biotinylated CD32b,and 2 rounds on biotinylated CD32b and 2 depletion rounds onbiotinylated CD32aR (clones denoted N3A, 03A, P3A, Q3A, P4B and Q4B).Selection was then performed as previously described.

2.5. Pool Cloning in the pMGM05 Vector:

The clones selected after the selection rounds were directly transferredin a mixture (about 10⁴ clones per condition) into the eukaryote vectorpMGM05-CD20 (pCEP4 InvitroGen), which contains the same cloning sites asthe pMG58 phagemid for the Fc fragment (BamHI and NotI) and the VHvariable chain of the anti-CD20 antibody. This construction leads tomutation of two amino acids in Fc (aa224 and 225, HT changed to GS) andaddition of the EFAAA sequence at the C-terminal of Fc, but allows rapidtesting of a very large number of clones. It was initially verified thatthese mutations do not modify the binding of IgG-WT to the differentreceptors. Thereafter, the positive controls were cloned in this systemfor validation thereof:

-   -   IgG1-5239D, 1332E, derived from the anti-CD19 XmAb5574 antibody        by Xencor (C1): positive control for CD16a;    -   IgG1-G236A, derived from Xencor (C4): positive control for        CD32aH/R;    -   IgG1-K326W, E333S, derived from Abgenix/Genentech (C3): positive        control for C1q; and    -   IgG1-5267E, L328F, derived from the anti-CD19 XmAb5574 antibody        by Xencor (C5): positive control for CD32b.

The DNA of about one hundred clones, isolated by selection rounds, weresequenced by PCR on colonies. After bioinformatic analyses, the clonescomprising new mutations were frozen at −80° C. in XL1-Blue bacteria andthe sequences included in our database. As a result, 158 clones wereisolated from the Mut3 bank, 371 clones from the Mut4sel bank and 171clones from the Mut5 bank.

2.6. Production of Variants in HEK293 Cells:

The light chain of anti-CD20 was inserted in a pCEP4 vector, identicalto the vector used for the heavy chain, denoted pMGM01-CDC20 (pCEP4InvitroGen). HEK293-F Freestyle™ cells (Invitrogen), cultured in 24-wellplates were co-transfected with the vectors pMGM01-CD20 and pMGM05-CD20(Fc-WT and variants) in equimolar amounts (250 ng/ml) with a FreestyleMAX reagent (1 μl/ml) following standard procedures (Invitrogen). Thecells were cultured in suspension in a serum-free medium for 7post-transfection days and the supernatants (1 ml) containing IgGs wereharvested after centrifugation of the cells at 100 g for 10 min. TheIgGs secreted in the supernatants were quantified using an ELISA assayon recombinant protein L (Pierce), with a purified anti-CD20 antibodyproduced in 293-F cells used as standard. The supernatants and standardantibodies diluted in series in PBS/0.05% Tween-20, were assayed onMaxisorp immunoplates (Nunc) previously coated with 0.25 μg proteinL/well and blocked with 5% skim milk in PBS. After incubation for 1 hourat 37° C., the wells were washed three times with PBS/0.05% Tween-20.Binding of IgG variants was detected with a F(ab′)2 fragment of goatanti-human IgG HRP (specific to the γ chain) (Sigma). The IgG variantsproduced were quantified (1-4 μg/ml) using the standard curve.

2.7. ELISA Assays on IgG Variants Produced in the Supernatants of 293-FCells:

The IgG variants were assayed by ELISA for their binding to the humanC1q complement and several human FcRs. Maxisorp immunoplates were coatedwith 0.5 μg C1q complement/well, 0.05 μg CD32aH/well, 0.2 μg CD16aF/wellor 0.1 μg CD16aV/well in PBS. Immobilising nickel chelating plates(Nunc) were coated with 0.1 μg CD32aR/well or 0.4 μg CD32b/well in 0.01M KCl. After coating overnight, at 4° C., the plates were washed twicewith PBS/0.05% Tween-20 and saturated with PBS/4% BSA for 2 hours at 37°C. In parallel, the supernatants were diluted in PBS to a finalconcentration of 0.5 μg IgG/m1 and mixed with F(ab′)2 fragments of goatanti-human IgG HRP at the same concentration for 2 hours at ambienttemperature. The IgGs aggregated to F(ab′)2 were then incubated undergentle agitation for 1 hour at 30° C. on saturated ELISA plates withoutdilution for C1q, CD16aF, CD32aR and CD32b (i.e. IgG at 0.5 μg/ml),diluted in PBS at 0.25 μg/ml for CD16aV and CD32aH. The plates were thenwith detected with TMB (Pierce) and absorbance read at 450 nm.

Selections on the Mut3 Bank:

By means of this ELISA assay, the variants selected by phage displaywere assayed for their binding to the C1q complement and to thedifferent receptors. They were assayed by comparison with the wild-typeFc (Fc-WT) and positive controls. 36 positive clones on CD16aV and/orC1q were thus selected on the Mut3 bank and used to construct the Mut4bank.

Selections on the Mut4sel Bank:

ELISA assays performed on the 371 isolated clones allowed identificationof 116 clones having a ratio higher than 2 for at least one FcγR and 17clones with a ratio higher than 3 for C1q alone, which thereforecorresponds to 133 clones of interest (Table 1).

TABLE 1 133 clones of interest isolated Name of Results of ELISA assaysmutant Mutations C1q CD16aV CD32aR CD32b K3B-01 N315D, N361H, P396L 3.462.01 7.65 7.30 A3A-105 G316D, K326E 5.18 2.48 4.50 7.13 G3A-59 N315D,T350A, P396L 4.05 2.22 7.95 6.19 J3B-81 V284L, V323I, P352S, 4.32 2.5213.17 6.12 A378V, Y436H J3A-123 V266M, P352S, A378V 0.77 0.35 7.76 5.93J3B-118 P396L, N421T 3.88 2.58 9.00 4.99 A3A-46 T350A, P396L 2.19 2.406.17 4.79 J3A-129 T350A, G385R, P396L 1.33 2.84 6.56 4.33 QL4A-55 N315D,P396L 2.06 2.00 3.41 4.27 A3A-27 K326T, H435R 5.81 2.21 3.41 4.10A3A-123 N315D, P396L, K447N 4.30 2.70 4.53 4.08 J3A-109 N315D, P396L,N434K 1.75 2.46 6.01 4.07 J3B-124 P396L 2.25 2.52 7.78 3.86 A3A-79E283G, A378T, V397M 1.62 1.35 3.07 3.83 G3A-69 K326T, V397M, F423L, 3.302.51 5.38 3.43 A431V A3A-178 P396L, F423S 4.03 1.73 3.55 3.37 DS3A-39V284L, A378V 2.76 3.18 3.48 3.30 J3A-89 V284L, N325S, A378V 0.80 0.514.50 3.29 DS3A-09 A378V 1.30 3.03 2.70 3.21 J3A-120 N315D, P343S, P396L1.66 2.32 4.71 3.16 K3A-35 K290E, S375R, F405V 1.89 1.64 3.93 3.12DL3A-58 S375R 1.85 2.25 3.95 3.11 A3A-50 V284L, K322E, A378V 0.67 1.505.08 3.10 DL4A-146 P352S, A378V 1.56 2.31 2.35 3.10 A3A-91 K340E, A378V,N389S, 2.39 1.88 2.67 3.03 N421T DS3A-93 F243I, P352S, A378V 1.29 1.714.19 2.97 A3A-184 K334N, P352S, V397M 1.44 2.90 3.06 2.91 A3A-112 T307P,N389T, V397M, 3.17 1.74 2.39 2.91 S442F A3A-132 K248E, N315D, P396L 5.032.12 3.70 2.79 DS3B-92 V284L, Y349H, A378V 2.52 1.98 2.57 2.79 QL3A-61N286I, A378V 1.33 2.07 2.20 2.77 J3B-115 T359A, S383R, V397M 1.72 1.695.17 2.77 A3A-173 K248E, K334R, A378V OVER 1.63 2.96 2.76 (>30) A3A-164A378V, K447N 1.64 2.41 2.82 2.71 G3A-118 N286I, P352S, A378V 1.21 2.454.83 2.68 QS5A-66 T394P 2.64 1.81 2.05 2.67 DL4A-55 A378V, N421T 1.201.87 2.34 2.66 G3A-176 V259A, V284L, A378V, 1.05 1.68 2.10 2.61 N421TG3A-09 A378T, V397M, K447N 1.58 2.23 3.12 2.45 DL3A-131 A378T, V397M1.20 1.72 2.30 2.40 J3A-10 T307P, P396L 5.18 2.74 5.71 2.39 J3B-49N315D, T393A, P396L 1.42 1.77 5.17 2.30 K3A-41 V302A, P352L, L398P 1.110.77 2.17 2.30 DL4A-54 P352L, Q418P 1.35 1.49 1.58 2.30 A3A-06 K248E,A378T, V397M 4.79 1.59 1.78 2.29 G3A-154 A378T, V397M, F423P, 2.51 2.114.14 2.28 S442P A3A-12 V363I, V397M 3.02 1.56 2.99 2.27 J3A-14 K326T1.83 1.43 4.25 2.26 G3A-05 V323I, S383N 2.21 1.33 3.37 2.25 J3B-138K334R, T394P 4.41 1.09 5.34 2.24 QL2A-11 V302A 0.97 0.68 1.52 2.22K3B-32 S254F, A378T, V397M 5.80 1.42 3.72 2.22 G3A-88 K320E, T394P,G402D 1.32 1.88 4.94 2.21 K3B-30 I253F, K326T, F423L, 7.34 1.58 5.992.20 A431V A3A-176 V284A, A378V, A431T 3.32 1.82 2.01 2.20 K3A-59 N315H,Y319H, V323I 2.88 0.59 3.33 2.17 J3B-120 A378V, L398P 1.29 1.67 2.942.17 QL4B-10 S383R, V397M 1.40 1.35 1.95 2.08 J3B-89 K290E, V3081,A327T, 0.50 0.14 0.30 2.05 S383N G3A-83 C226Y, A378V, N421T 0.90 2.262.00 2.04 G3A-165 V323I, T393I 1.33 1.52 4.15 2.04 J3B-135 K248E, S383R,V397M, 6.36 1.51 3.99 2.04 N434S A3A-137 K290E, V323I, F405L 1.91 1.653.02 2.03 K3B-94 R255W, A287T, P352S, 4.66 1.52 5.08 2.02 A378V A3A-30V284L, T350A 2.09 1.47 2.47 2.01 K3A-07 N315D, A378V 1.30 1.76 2.26 2.01G3A-164 T394P, N434S 1.72 1.19 2.29 1.99 J3A-16 N286Y, P352S, A378V 1.461.83 3.54 1.98 G3A-43 A231V, A378V 1.13 2.44 3.57 1.96 A3A-09 V308G,V323I 3.65 1.23 2.26 1.95 G3A-106 S254F, V284L, A378V 1.57 1.95 3.961.94 J3A-08 K274R, A378V, N421T 1.17 1.53 3.47 1.94 QL3A-20 V397M 2.491.96 2.60 1.93 G3A-108 T394P, K414R 1.50 1.54 3.67 1.91 G3A-163 V323I,M428T 1.06 1.05 2.41 1.89 A3A-125 T394P, K447N 1.94 1.83 2.48 1.84J3B-109 E345G, V397M 1.18 1.36 2.37 1.83 DS3B-33 K326T, F423L, A431V5.29 1.31 1.73 1.80 QL4A-28 F243L, P247T, Q362R, 0.74 1.65 2.88 1.80G402D, S440N J3B-101 Y278H, N315D, P396L 1.18 1.64 2.35 1.78 A3A-140D312G, A378T, V397M 1.47 1.39 2.23 1.76 G3A-25 V262A, V305A, A378V 1.761.56 2.01 1.70 G3A-45 K246R, A378V 1.15 2.46 2.76 1.68 QS6A-78 V323I1.41 1.31 2.03 1.68 K3B-91 V3081, K326T, F423L, 4.84 1.13 2.71 1.65A431V G3A-103 K248E, A378V 2.54 2.00 1.96 1.64 G3A-07 N276S, T394P,K439Q 2.27 1.13 2.17 1.59 A3A-17 K290E, E380G 1.64 2.02 1.91 1.59 J3B-16S267G, A378T, V397M 0.85 0.57 4.95 1.58 J3B-23 N286I, A378V, F423Y 1.131.81 3.33 1.57 J3B-68 K320E, T350A 0.82 1.57 3.30 1.55 A3A-37 A231T,K290E, S383N, 1.41 1.36 2.02 1.53 F423L J3A-49 K320R, A378T, V397M 0.600.45 2.15 1.52 A3A-31 K334R, L410R 6.23 1.36 1.57 1.50 A3A-41 V323I,P352L, L398P 2.00 1.46 2.53 1.47 K3A-36 K248E, K320M 2.81 1.69 2.34 1.42J3A-06 A378T, V397M, V412M 1.60 1.65 2.74 1.41 G3A-139 T307N, V323I 1.301.10 2.57 1.41 G3A-98 S375R, N434S 1.14 1.56 3.02 1.40 K3B-80 V284L,T366A, A378V 2.58 1.25 2.27 1.39 J3B-74 P230S, N389S, T394P 0.94 0.651.81 1.39 K3B-41 Y349S, V397M 1.82 1.27 2.11 1.38 G3A-13 K248E, A339T,T350A, 2.10 1.28 2.89 1.37 S440N J3B-44 K246E, K274E, V397M 1.59 1.052.38 1.37 K3B-43 A231P, K290E, S383N 1.66 1.43 2.15 1.36 J3B-107 T307P,A378T, N389T 1.50 1.32 2.05 1.36 K3B-33 K334R, K392R, S440G 4.87 0.770.97 1.33 K3A-11 K246E, K290E, T307P, 1.73 1.65 2.00 1.33 N389T K3B-49D376G, S383R, V397M 2.54 1.34 2.57 1.33 G3A-95 I336T, A378V 1.05 2.251.82 1.31 K3B-90 V308A, K334R, A378V, 1.32 1.30 3.72 1.27 K447N A3A-146K290E, R355Q, S383N 1.49 1.38 2.15 1.26 A3A-11 S254F, K447N 4.78 1.181.02 1.22 G3A-31 K248E, K334Q 3.60 1.52 1.06 1.21 QL2A-16 K248E, N421T5.30 0.79 0.77 1.19 J3A-43 K248E, P352L, Q418P 2.96 1.23 2.13 1.19J3A-28 E333G, A378T, V397M 1.89 1.83 1.73 1.18 A3A-07 L365P, T366S,A378T 1.20 1.94 1.17 1.18 A3A-96 K248E, L365P, A378T 4.09 1.43 0.70 1.12K3B-87 V323I, F405L 1.56 0.93 2.36 1.10 G3A-148 K248E, S375R 3.79 1.251.64 1.08 K3B-34 K334N, T394P, S408N, 0.62 1.18 2.25 1.02 K414N K3B-89L309M, A378V 1.98 1.42 2.07 1.02 G3A-49 T307A, E380G 3.66 0.91 0.82 1.02G3A-159 E382G, L432P, 3.00 0.91 1.13 0.98 Q438DEL G3A-28 T307P, H435R3.49 1.06 0.95 0.96 K3B-78 H310R, P352L, Q418P 1.20 0.84 2.04 0.96G3A-22 P352S, E382G, L432P, 3.11 0.95 0.62 0.94 Y436N G3A-56 K248E,N421T, K447N 3.25 0.71 1.03 0.89 G3A-145 E430G, K447N 4.34 1.01 0.810.88 A3A-21 S267N, N384D, N389S, 4.32 1.00 0.56 0.88 P396L G3A-33 K248E,S267N, 1336M, 5.67 0.79 0.39 0.76 P352L, P396L G3A-51 V240I, K246E,P353L 3.90 0.95 0.51 0.64

Among these clones, 47 improved clones were used to construct the Mut5bank.

From these results, 18 new variants were constructed by directedmutagenesis so as to accumulate mutations of interest (Table 2).

TABLE 2 18 new variants constructed and compared with reference variantsNA = Non-Determined Name of Results of ELISA assays mutant Mutations C1qCD16aF CD16aV CD32aH CD32aR CD32b A3A-105 G316D, K326E 5.18 1.31 2.481.50 4.50 7.13 A3A-105A G316D, K326E, 15.33 1.09 1.45 0.66 5.60 1.21T394P A3A-105B G316D, K326E, 14.30 2.25 3.52 3.92 12.64 4.16 P396LA3A-105C G316D, K326E, 9.61 2.90 1.86 1.81 11.00 2.22 V397M A3A-105DG316D, K326E, 13.32 2.46 3.68 1.39 8.20 5.96 A378V A3A-184 K334N, P352S,1.44 1.71 2.90 1.34 3.06 2.91 V397M A3A-184A K334N, P352S, 1.75 3.404.74 3.25 5.03 2.32 A378V, V397M DS3A-09 A378V 1.30 3.15 3.03 2.90 2.703.21 DS3A-09A A378V, T394P 1.24 2.29 2.83 2.26 2.00 1.26 DS3A-09B A378V,P396L NA 1.83 2.43 NA NA NA DS3A-09C A378V, V397M NA 1.48 2.25 NA NA NAG3A-43 A231V, A378V 1.13 1.79 2.44 1.42 3.57 1.96 G3A-43A A231V, A378V,NA 1.13 1.65 NA NA NA T394P G3A-43B A231V, A378V, NA 1.85 2.42 NA NA NAP396L G3A-43C A231V, A378V, NA 1.81 2.68 NA NA NA V397M G3A-45 K246R,A378V 1.15 1.92 2.46 1.38 2.76 1.68 G3A-45A K246R, A378V, NA 1.34 2.34NA NA NA T394P G3A-45B K246R, A378V, NA 1.37 1.75 NA NA NA P396L G3A-45CK246R, A378V, NA 1.51 2.21 NA NA NA V397M G3A-95 I336T, A378V 1.05 1.292.25 1.17 1.82 1.31 G3A-95A I336T, A378V, NA 1.50 1.90 NA NA NA T394PG3A-95B I336T, A378V, NA 2.00 2.54 NA NA NA P396L G3A-95C I336T, A378V,NA 1.55 2.01 NA NA NA V397M J3B-118 P396L, N421T 3.88 1.26 2.58 NA 9.004.99 J3B-118A A378V, P396L, NA 1.76 3.37 NA NA NA N421T QL3A-20 V397M2.49 0.62 1.96 1.09 2.60 1.93 QS5A-66 T394P 2.64 NA 1.81 NA 2.05 2.67

Finally, from these 151 assayed variants, 26 variants of interest wereselected for production on a larger scale and more specific study (Table3).

TABLE 3 26 selected new variants NA = Non-Determined Name of Results ofELISA assays mutant Mutations C1q CD16aF CD16aV CD32aH CD32aR CD32bA3A-105 G316D, K326E 5.18 1.31 2.48 1.50 4.50 7.13 A3A-105D G316D,K326E, 13.32 2.46 3.68 1.39 8.20 5.96 A378V A3A-14 S298N, A378V 0.530.96 0.85 0.22 0.35 0.88 A3A-173 K248E, K334R, NA NA 1.63 NA 2.96 2.76A378V A3A-184 K334N, P352S, 1.44 1.71 2.90 1.34 3.06 2.91 V397M A3A-184AK334N, P352S, 1.75 3.40 4.74 3.25 5.03 2.32 A378V, V397M A3A-31 K334R,L410R 6.23 NA 1.36 NA 1.57 1.50 DL4A-54 P352L, Q418P 1.35 1.18 1.49 1.091.58 2.30 G3A-103 K248E, A378V 2.54 1.17 2.00 0.83 1.96 1.64 G3A-139T307N, V323I 1.30 0.81 1.10 0.65 2.57 1.41 G3A-43 A231V, A378V 1.13 1.792.44 1.42 3.57 1.96 G3A-45 K246R, A378V 1.15 1.92 2.46 1.38 2.76 1.68G3A-88 K320E, T394P, 1.32 NA 1.88 NA 4.94 2.21 G402D G3A-95 I336T, A378V1.05 1.29 2.25 1.17 1.82 1.31 J3A-06 A378T, V397M, 1.60 1.24 1.65 1.582.74 1.41 V412M J3A-14 K326T 1.83 0.52 1.43 0.99 4.25 2.26 J3A-16 N286Y,P352S, 1.46 1.35 1.83 1.77 3.54 1.98 A378V J3A-28 E333G, A378T, 1.891.41 1.83 1.39 1.73 1.18 V397M J3B-115 T359A, S383R, 1.72 1.32 1.69 1.425.17 2.77 V397M J3B-118 P396L, N421T 3.88 1.26 2.58 NA 9.00 4.99J3B-118A A378V, P396L, NA 1.76 3.37 NA NA NA N421T J3B-16 S267G, A378T,0.85 0.59 0.57 0.42 4.95 1.58 V397M J3B-23 N286I, A378V, 1.13 1.91 1.811.57 3.33 1.57 F423Y K3B-01 N315D, N361H, 3.46 0.98 2.01 2.10 7.65 7.30P396L K3B-90 V308A, K334R, 1.32 1.06 1.30 1.40 3.72 1.27 A378V, K447NQL2A-16 K248E, N421T 5.30 0.69 0.79 0.33 0.77 1.19

Selections on the Mut5 Bank:

The ELISA assays performed on the 171 isolated clones allowed theidentification of 87 clones with a ratio higher than 2 for at least oneof the assayed FcγRs or C1q complement (Table 4).

TABLE 4 87 clones with a ratio higher than 2 Name of Results of ELISAassays mutant Mutations C1q CD16aF CD16aV CD32aH CD32aR CD32b P3A-01K334N, S383R 0.93 1.34 2.05 1.10 2.00 1.57 P3A-17 K334N, Y373D, 0.881.46 1.63 1.35 2.17 1.55 A378V P3A-18 K248E, N286Y, 24.77 0.94 1.62 1.071.24 1.13 K334R, A378V, K447N P3A-23 N361H, P396L 2.70 1.75 2.49 2.533.33 1.76 P3A-30 K288E, K334N, 1.18 1.35 4.33 2.14 1.79 1.41 P352S,V397M P3A-31 K248E, T359A, 5.80 0.80 2.05 1.80 1.62 1.51 S383R, V397MP3A-50 T307P, A378V 1.11 1.55 2.89 2.20 1.85 1.44 P3A-56 G316D 1.74 1.071.90 2.02 1.45 1.39 P3A-70 K274R, A378V, 1.13 1.93 4.05 3.54 4.89 3.20V397M P3A-74 V302A, V397M 0.92 0.72 0.63 1.47 2.43 1.32 P3A-94 L309P,T359A, 1.02 1.32 2.11 2.54 1.79 1.32 S383R, V397M N3A-111 V302A, K334N,1.36 0.82 1.18 1.20 2.73 2.33 S375R N3A-113 E283G, Y349S, 6.55 1.30 1.732.17 5.19 2.94 P396L N3A-114 K320E, S375R, 0.69 1.03 1.12 0.92 2.11 1.60F405V N3A-117 K334N 1.32 1.61 2.28 1.31 1.93 1.98 N3A-123 K326E 3.151.03 2.21 1.27 7.71 3.53 N3A-132 S375R, N389S 1.71 0.99 1.10 1.41 3.502.35 N3A-133 K334N, P352S, 0.77 1.02 2.23 0.88 1.25 1.40 K447N N3A-138S298T, K334R, 3.56 0.90 0.68 0.59 0.73 1.17 K370E N3A-14 T307N, A378V,1.00 1.70 2.83 2.50 2.49 1.74 K414R N3A-141 T350A, V379A, 2.82 1.30 1.792.36 5.60 5.70 G385R, P396L N3A-145 S267G, K290E, 0.99 0.70 0.38 0.312.45 1.33 E293G, E380G, V397M N3A-150 S267G, K334N, 0.86 0.76 0.68 0.334.82 1.78 P352S, V397M N3A-161 K248E, N315D, 8.59 1.28 2.49 1.12 1.231.32 A378V N3A-175 S375R, F405V 0.94 1.16 2.47 1.99 1.30 1.10 N3A-177V282A, K334R, 5.95 1.15 1.28 1.68 3.22 1.68 T394P N3A-190 T307P, T394P2.46 1.35 2.46 2.93 3.33 1.94 N3A-27 K290E, A378V, 1.67 2.25 3.77 2.052.59 1.49 K392R, S440G N3A-28 T366A, A378V 1.04 1.77 2.89 2.26 1.84 1.36N3A-32 K326T, K334N, 1.61 2.47 4.35 1.59 3.24 2.04 P352S, N421T N3A-52K326T, S383R, 3.70 1.11 2.07 1.92 5.40 2.41 V397M N3A-58 V284L, K334R,5.37 1.02 1.67 2.01 4.00 1.67 A378V, K447N N3A-59 N315D, T366A, 1.081.25 1.66 1.75 2.73 1.56 G385R, P396L N3A-61 V308A, K334R, 2.07 1.101.57 1.11 2.87 2.67 A378V N3A-74 K288R, T394P 1.38 1.22 1.51 1.58 2.151.73 N3A-85 K334R, V397M 4.96 1.11 1.45 1.89 3.75 2.77 N3A-87 K290E,K320E, 0.91 2.15 2.85 3.12 5.94 4.63 T350A, P396L N3A-93 A378T, P396L1.63 1.84 2.21 2.55 3.86 3.06 O3A-04 A231V, T359A, 1.00 1.16 1.21 1.692.35 1.09 S383R, V397M O3A-05 K290E, T366A, 0.99 2.79 3.12 2.07 3.341.34 A378V O3A-10 Y300H, T394P 0.98 0.76 0.90 1.81 2.25 1.17 O3A-11R255W, A287T, 1.85 1.01 2.10 2.20 3.15 1.5 P352S, A378V, N421T O3A-16K334R, A378V, 4.30 1.71 2.51 2.43 3.98 1.59 K447N O3A-17 I336V, T359A,0.85 1.79 2.70 2.30 1.73 0.93 S383R, V397M O3A-22 K334R, P396L, 23.551.68 2.16 2.75 4.79 1.62 H435R O3A-24 K326T, K447N 2.12 1.23 2.27 1.552.94 1.40 O3A-25 V308A, K334R, 11.12 1.40 2.07 2.04 4.16 1.96 A378T,V397M O3A-34 A231V, Y349S, 0.73 1.40 1.90 2.34 2.62 1.28 V397M O3A-38K248E, N286Y, 2.17 1.59 1.16 0.99 0.69 0.57 Q418P O3A-40 V308A, A378T,0.82 1.65 2.15 2.09 2.64 1.03 V397M, V412M O3A-42 S304N, A378V 1.04 2.160.53 0.87 0.87 1.77 O3A-44 A378V, K439R 1.95 2.44 4.40 3.20 3.13 1.81O3A-45 D270N, K334R, 3.32 1.57 0.61 0.56 1.19 1.36 L410R O3A-46 V302A,A378V 0.94 1.15 1.75 2.36 2.91 1.37 O3A-50 K334R, A378V, 3.62 1.28 2.492.15 4.02 2.30 N421T O3A-57 T359A, V397M 1.50 1.18 2.27 2.23 2.78 1.59O3A-66 A378T, T394P 1.10 1.42 2.42 2.36 2.45 1.76 O3A-67 K248E, N361H2.05 0.89 1.36 1.06 1.07 1.76 O3A-69 K290E, A378V 1.44 2.64 4.04 2.624.11 2.06 O3A-80 K274R, T394P, 1.26 1.26 1.81 2.16 2.32 1.28 G402D,K447N O3A-81 K274R, A378T, 1.36 1.20 2.39 2.06 3.08 1.58 V397M O3A-86K248E, K290E, 24.34 2.49 4.89 2.86 4.19 2.57 N361H, P396L O3A-93 K290E,T394P 2.07 1.45 3.53 2.54 3.36 1.78 P4B-89 T307P, T366A, 0.63 2.99 4.150.70 1.19 0.87 A378V Q4B-08 V284L, K290E, 2.43 1.75 3.47 2.69 3.58 1.63A378V Q4B-15 K274R, A378V 1.26 1.04 2.69 2.61 1.75 1.03 Q4B-18 I377T,A378V, 1.21 1.08 2.82 1.96 1.37 1.07 F423Y Q4B-34 K248E, K290M, 4.470.63 0.56 0.38 0.70 0.75 V308A, P352S Q4B-59 T350A, A378T, 1.32 2.222.01 2.76 2.52 1.51 V397I Q4B-61 K320E, T394P, 0.87 1.10 0.98 1.60 2.431.08 V397M Q4B-68 T307P, A378V, 3.33 1.53 4.12 4.01 3.69 1.03 T394PQ4B-91 K334R, A378V 2.81 1.14 2.35 2.61 2.46 0.94 Q3A-01 P352L, A378V1.13 1.14 1.53 2.04 1.81 0.87 Q3A-39 N286I, P396L, 2.80 1.63 1.99 3.523.09 1.02 N421T Q3A-58 S267G, V397M 0.73 1.30 0.34 0.40 2.87 1.02 Q3A-85P396L, N421T, 3.80 1.65 2.17 3.67 3.50 1.13 K447N O3A-103 K290E, K320T,2.11 1.22 2.08 2.76 2.41 1.50 A378V O3A-117 K334R, T394P, 0.98 1.05 1.712.29 3.05 1.37 N421S O3A-119 K334N, V397M 1.53 1.04 1.97 1.45 2.28 1.18O3A-126 P227S, V284L, 4.58 1.31 2.93 2.85 4.47 1.29 A378V O3A-127 V302A,K334R, 1.53 1.00 1.24 2.85 3.56 1.37 T366A, S383R, V397M O3A-131 C261R,A378T, 2.25 1.03 1.72 2.00 2.27 1.15 V397M, V412M O3A-137 K248E, T350A0.84 0.66 0.43 0.68 2.06 0.97 O3A-172 K320E, T394P 8.19 0.88 1.46 2.012.52 1.06 O3A-179 R255Q, G385R 0.81 0.74 0.83 0.57 2.86 1.30 O3A-186T307P, V397M 1.09 1.41 2.32 2.17 3.97 1.48 O3A-99 N276S, N286I, 2.141.32 2.27 2.84 3.09 1.42 T359A, S383R, V397M

Among these variants, 10 variants of interest were selected (Table 5).

TABLE 5 10 variants Name of Results of ELISA assays mutant Mutations C1qCD16aF CD16aV CD32aH CD32aR CD32b O3A-05 K290E, T366A, 0.99 2.79 3.122.07 3.34 1.34 A378V P4B-89 T307P, T366A, 0.63 2.99 4.15 0.70 1.19 0.87A378V O3A-86 K248E, K290E, 24.34 2.49 4.89 2.86 4.19 2.57 N361H, P396LN3A-32 K326T, K334N, 1.61 2.47 4.35 1.59 3.24 2.04 P352S, N421T O3A-44A378V, K439R 1.95 2.44 4.40 3.20 3.13 1.81 N3A-27 K290E, A378V, 1.672.25 3.77 2.05 2.59 1.49 K392R, S440G N3A-87 K290E, K320E, 0.91 2.152.85 3.12 5.94 4.63 T350A, P396L Q4B-68 T307P, A378V, 3.33 1.53 4.124.01 3.69 1.03 T394P N3A-58 V284L, K334R, 5.37 1.02 1.67 2.01 4.00 1.67A378V, K447N Q3A-39 N286I, P396L, 2.80 1.63 1.99 3.52 3.09 1.02 N421T

3. Production and Purification of Variants of Interest

The IgG variants were obtained by direct mutagenesis in pCEP4-WT-H-CD20.The IgG controls, i.e. C1, C3, C4, C5 and wild-type (WT), were producedwith the G1m3 allotype (comprising 3 mutations compared with G1 m1,17:(K214R/)D356E/L358M).

The 26 IgG variants derived from the Mut4sel bank, and the wild-type,were produced with the G1 m1,17 allotype. They were produced byincubation for 6-7 days in batches (250-300 ml) of 293-E cells(Freestyle Invitrogen) in F17 medium.

Centrifugation and filtration were then carried out.

Purification was performed on Protein A Hi-Trap, and elution with a 25mM citrate buffer, pH=3.0, neutralisation and dialysis in TBS or PBSbefore sterilisation.

10 mg of each IgG control, i.e. C1, C3, C4, C5 and wild-type (WTG1m3 andWTG1 m1,17) were obtained, and 2-3 mg of 26 IgG variants.

Their characterization shows that the molecular weight is maintained andthat glycosylation profiles are similar for all variants.

4. Assays of Variants of Interest

4.1. Binding Assays on FcRn

Jurkat-FcRn cells were incubated at pH=6.0 with the IgG variants atdifferent concentrations (0 to 1000 μg/ml) and with Rituximab-Alexa.

Flow cytometry was conducted on the bound Rituximab-Alexa.

The results do not show any loss of binding to FcRn for all IgGvariants.

4.2 Binding Assays to the Antigen

Raji cells were incubated with the IgG variants at 1 μg/ml for 15minutes at 4° C.

The bound IgGs were detected by binding with a PE anti-human IgGsecondary antibody (for 15 minutes at 4° C.).

The results show that recognition of the antigen is not deteriorated bythe different mutations on the Fc.

All the IgG variants were bound to CD20 on the cells, similar to theIgG-WT control.

4.3 ELISA Binding Assays to CD16aV/F

The purified antibodies were assayed with ELISA for binding to CD16F andCD16aV following the same protocol as described under item 2.6, dilutingthe antibodies to different concentrations.

4.4. ADCC Activity Assays

NK cells were incubated with target Raji cells expressing CD20, in thepresence of different concentrations of IgG variants (0.005 to 5000ng/ml).

The level of intracellular LDH released by the lysed target cells wasmeasured.

Human NK cells were purified from the peripheral blood of healthyvolunteer donors using the negative depletion technique developed byMiltenyi. The ADCC assay comprised the incubation of NK cells withtarget Raji cells expressing the CD20 antigen, in the presence ofdifferent concentrations of anti-CD20 antibodies. After an incubationtime of 16 hours, the cytotoxicity induced by the anti-CD20 antibodieswas measured by quantifying intracellular lactate dehydrogenase (LDH) inthe cell supernatants. The results of specific lysis are expressed as alysis percentage as a function of antibody concentration. The EC50 value(antibody concentration inducing 50% of the maximum lysis induced byIgG-WT) and Emax value (percentage maximum lysis) were calculated usingthe software GraphPad PRISM.

The results are given in Tables 6 and 7.

TABLE 6 Results of ADCC assays Mutant G3A43 G3A45 J3B-118 J3B16 A3A-184WT EC50: 1.03 4.38 1.27 >5545.36 2.35 12.34 Antibody concentration(ng/ml) giving 50% lysis of an internal control Ratio [WT]/[antibody]11.98 2.82 9.72 <0.0022 5.25 1

TABLE 7a Results of ADCC assays EC50: Antibody concentration (ng/ml)giving 50% maximum lysis of Ratio Mutant an internal control[WT]/[antibody] G3A-103 0.31 33.90 A3A-184A 0.34 30.91 J3B-23 0.57 18.44J3A-28 0.57 18.44 K3B-01 0.69 15.23 A3A-14 0.82 12.82 G3A-95 0.97 10.84J3B-118A 0.97 10.84 J3A-16 1.27 8.28 J3A-06 1.27 8.28 A3A-105D 1.27 8.28G3A-139 2.80 3.75 QL2A-16 4.36 2.41 J3A-14 7.39 1.42 G3A-88 8.07 1.30 WT10.51 1 K3B-87 16.33 0.64 DL4A-54 >5000 <0.002 A3A-90 >5000 <0.002A3A-34 >5000 <0.002

TABLE 7b Results of ADCC assays EC50: Antibody concentration (ng/ml)giving 50% maximum lysis of an Mutant internal control Ratio[WT]/[antibody] WT 0.45 1 A3A-173 1.38 0.33 K3B-90 0.75 0.60 A3A-1051.374 0.33 A3A-31 1.839 0.24 J3B-115 0.57 0.79

TABLE 7c Results of ADCC assays (EC45 instead of EC50) EC45: Antibodyconcentration (ng/ml) giving 45% maximum lysis of an Mutant internalcontrol Ratio [WT]/[antibody] WT 1.60 1 O3A-44 2.6 0.62 N3A-27 18.7 0.86N3A-58 4.4 0.36 O3A-86 4.4 0.36 N3A-87 1.0 1 P4B-89 3.6 0.44 O3A-05 0.28 N3A-32 2.9 0.55 Q3A-39 0.3 5.33 Q4B-68 1.7 0.94 The best variants areA3A-184A et G3A-103.

4.5. CDC Activity Assays

Target Raji cells expressing the CD20 antigen were incubated withdifferent concentrations of anti-CD20 antibodies (0 to 5000 ng/ml) inthe presence of rabbit serum as complement source (Cedarlane, 1/10dilution). After an incubation time of 1 hour at 37° C., the level ofLDH released into the supernatant by the lysed target cells was measuredchromogenically (cytotoxicity detection kit by Roche Applied Sciences)and used to quantify antibody-mediated complement-dependentcytotoxicity. The results are expressed as percent lysis. EC50 (numberof antibodies inducing 50% maximum lysis) and Emax (percent maximumlysis) were calculated using the software GraphPad PRISM.

The results are given in Tables 8 and 9.

TABLE 8 Results of CDC assays Antibody concentration (ng/ml) giving 50%lysis Antibody of an internal control Ratio [WT]/[antibody] A3A-17316.01 16.36 K3B-90 23.12 11.33 J3B-118 38.69 6.77 A3A-105 64.76 4.05A3A-31 108.37 2.42 J3B-115 145.44 1.80 G3A-43 168.49 1.55 G3A-45 195.201.34 A3A-184 210.10 1.25 J3B-16 210.10 1.25 WT 261.97 1

TABLE 9a Results of CDC assays Antibody concentration (ng/ml) giving 50%lysis of an Antibody internal control Ratio [WT]/[antibody] J3B-118A21.564 10.98 J3A-28 31.004 7.64 G3A-103 33.339 7.10 A3A-105D 38.550 6.14QL2A-16 51.544 4.59 K3B-01 59.600 3.97 J3A-06 64.089 6.70 J3A-14 64.0896.70 A3A-184A 74.107 3.20 G3A-139 85.691 2.76 J3B-23 99.085 2.39 K3B-87106.548 2.22 A3A-34 123.202 1.92 J3A-16 132.481 1.79 DL4A-54 164.7271.44 G3A-95 177.134 1.34 WT 236.837 1 G3A-88 273.856 0.86 A3A-14 340.5130.70 A3A-90 566.098 0.42

TABLE 9b Results of CDC assays Antibody concentration (ng/ml) giving 50%lysis of an Antibody internal control Ratio [WT]/[antibody] WT 133 1O3A-44 129 1.03 N3A-27 179 0.74 N3A-58 36 3.69 O3A-86 44 3.02 N3A-87 1061.25 P4B-89 115 1.16 O3A-05 91 1.46 N3A-32 66 2.02 Q3A-39 61 2.18 Q4B-68126 1.06

Table 10 below gives the results obtained with some variants classifiedinto sub-groups.

TABLE 10 ELISA ratio (purified antibodies) Name Mutations ADCC CDCCD32aH CD32aR CD32b CD64 G3A-103 K248E, A378V 33.9 7.1 0.5 0.9 1.2 1.2J3A-28 E333G, A378T, V397M 18.4 7.6 1.1 1.3 1.6 1.3 J3B-118A P396L,N421T, A378V 10.8 11.0 2.7 1.8 3.7 1.4 J3B-118 P396L, N421T 9.7 12.2 1.01.1 1.0 1.6 A3A-105D G316D, K326E, A378V 8.3 6.1 1.4 2.6 2.0 1.5 A3A-14S298N, A378V 12.8 0.7 1.1 0.8 0.9 1.6 G3A-95 I336T, A378V 10.8 1.3 0.70.9 1.0 1.3 A3A-184A K334N, P352S, V397M, A378V 30.9 3.2 1.3 4.3 3.1 1.5J3B-23 N286I, A378V, F423Y 18.4 2.4 ND ND ND ND K3B-01 N315D, N361H,P396L 15.2 4.0 5.7 1.0 1.0 2.3 G3A-43 A231V, A378V 12.0 2.8 5.3 3.6 2.71.4 J3A-06 A378T, V397M, V412M 8.3 3.7 0.84 2.0 2.4 1.3 J3A-16 N286Y,P352S, A378V 8.3 1.8 2.61 1.7 1.7 1.3 O3A-05 K290E, T366A, A378V 8.0 1.54.02 3.3 4.5 1.2 Q3A-39 N286I, P396L, N421T 5.3 2.2 4.83 2.3 3.8 1.5A3A-184 K334N, P352S, V397M 5.3 2.2 1.33 3.2 3.3 1.4 K3B-90 V308A,K334R, A378V, K447N 0.6 20.4 1.20 1.2 1.1 1.5 A3A-173 K248E, K334R,A378V 0.3 29.5 0.95 1.3 1.3 1.2 J3A-14 K326T 1.4 3.7 1.86 1.3 1.5 2.3G3A-88 K320E, T394P, G402D 1.3 0.9 2.94 3.7 3.9 2.4 N3A-87 K290E, K320E,T350A, P396L 1.0 1.3 3.92 3.7 9.0 1.5 J3B-115 T359A, S383R, V397M 0.83.2 4.65 5.1 4.2 1.5

Following Table 11 gives some possible variants of the invention:

TABLE 11 Starting Added Name Mutations variant mutation G3A-95D S298N,I336T, A378V G3A-95 S298N K3B-90A K248E, V308A, K334R, A378V, K3B-90K248E K447N G3A-88A K320E, K326T, T394P, G402D G3A-88 K326T N3A-87AK290E, K320E, K326T, T350A, N3A-87 K326T P396L A3A-105E K248E, G316D,K326E, A378V A3A-105D K248E A3A-14A K248E, S298N, A378V A3A-14 K248EA3A-184B K248E, K334N, P352S, V397M, A3A-184 K248E A378V G3A-43D K248E,A231V, A378V G3A-43 K248E G3A-95E K248E, I336T, A378V G3A-95 K248EJ3A-28A K248E, E333G, A378T, V397M J3A-28 K248E J3B-118B K248E, P396L,N421T, A378V J3B-118A K248E J3B-23A N286I, A378V, F423Y J3B-23 K248EK3B-01A N315D, N361H, P396L K3B-01 K248E A3A-105F G316D, K326E, E333G,A378V A3A-105 E333G A3A-14B S298N, E333G, A378V A3A-14 E333G A3A-184CE333G, K334N, P352S, V397M, A3A-184 E333G A378V G3A-103A K248E, E333G,A378V G3A-103 E333G G3A-43E A231V, E333G, A378V G3A-43 E333G G3A-95FE333G, 1336T, A378V G3A-95 E333G J3B-118C E333G, P396L, N421T, A378VJ3B-118 E333G J3B-23B N286I, E333G, A378V, F423Y J3B-23 E333G K3B-01BN315D, E333G, N361H, P396L K3B-01 E333G A3A-105G G316D, K326E, A378V,F423Y A3A-105 F423Y A3A-14C E333G, S298N, A378V, F423Y A3A-14 F423YA3A-184D K334N, P352S, V397M, A378V, A3A-184 F423Y F423Y G3A-103B K248E,A378V, F423Y G3A-103 F423Y G3A-43F A231V, A378V, F423Y G3A-43 F423YG3A-95G 1336T, A378V, F423Y G3A-95 F423Y J3A-28B E333G, A378T, V397M,F423Y J3A-28 F423Y J3B-118D P396L, N421T, A378V, F423Y J3B-118 F423YK3B-01C N315D, N361H, P396L, F423Y K3B-01 F423Y

1. Polypeptide comprising a mutated Fc region and having functionalactivity. mediated by the Fc region, that is modified compared with thatof a parent polypeptide, wherein said Fc region comprises at least onecombination of 2 mutations, said combination being selected from among:(i) one mutation selected from among 307N, 326E, 326T, 334N, 334R, 352L,378V, 378T, 394P, 396L, 397M, 421T; and (ii) at least one mutationselected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R,246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N,267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E,288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D,316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T,339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I,366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T,392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M,414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S,435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, the numbering beingthat of the EU Index or Kabat equivalent, and provided that mutation (i)does not take place on the same amino acid as mutation (ii). 2.Polypeptide according to claim 1, wherein the mutation (i) is selectedfrom among 378V, 396L and 397M.
 3. Polypeptide according to claim 2,further comprising a mutation selected from among 248E, 326T, 333G and423Y.
 4. Polypeptide according to claim 1, wherein said functionalactivity mediated by the Fc region is selected from amongantibody-dependent cell cytotoxicity (ADCC), complement-dependentcytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), and acombination of at least two of these activities.
 5. Polypeptideaccording to claim 1, having functional activity mediated by the Fcregion that is increased compared with that of the parent polypeptide,preferably by a ratio of at least 2, preferably higher than 5,preferably higher than 10, preferably higher than 15, preferably higherthan 20, preferably higher than 25, preferably higher than
 30. 6.Polypeptide according to claim 1, having functional activity mediated bythe Fc region that is reduced compared with that of the parentpolypeptide, preferably by a ratio of at least 2, preferably higher than5, preferably higher than 10, preferably higher than 15, preferablyhigher than 20, preferably higher than 25, preferably higher than
 30. 7.Polypeptide according to claim 1, wherein said mutated Fc region hasmodified affinity for at least one of the receptors (FcRs) of the Fcregion selected from among the C1q complement and FcgRIIIa (CD16a),FcgRIIa (CD32a) and FcgRIIb (CD32b) receptors.
 8. Polypeptide accordingto claim 1, wherein said mutated Fc region comprises 2 to mutationscompared with the parent polypeptide, preferably 2 to 10 mutations. 9.Polypeptide according to claim 1, wherein said mutated Fc region hasimproved affinity for the C1q complement, and comprises at least onecombination of 2 mutations, said combination comprising: i) one mutationselected from among 378V, 378T, 396L, 421T, 334R and 326E; and ii) atleast one mutation selected from among 361H, 290E, 316D, 248E, 410R,421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 286I, 315D and397M, the numbering being that of the EU Index or Kabat equivalent, andprovided that mutation (i) does not take place on the same amino acid asmutation (ii).
 10. Polypeptide according to claim 1, wherein saidmutated Fc region has improved affinity for the FcgRIIIa receptor(CD16a), and comprises at least one combination of 2 mutations, saidcombination comprising: i) one mutation selected from among 378V, 326E,397M, 334N and 396L; and ii) at least one mutation selected from among316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E,394P, 307P, 378V, 378T, 286I, 286Y and 298N, the numbering being that ofthe EU Index or Kabat equivalent, and provided that mutation (i) doesnot take place on the same amino acid as mutation (ii).
 11. Polypeptideaccording to claim 1, wherein said mutated Fc region has increasedaffinity for the FcgRIIa receptor (CD32a), and comprises at least onecombination of 2 mutations, said combination comprising: i) one mutationselected from among 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N;and ii) at least one mutation selected from among: 316D, 334R, 334N,323I, 231V, 246R, 336T, 378T, 286Y, 286I, 352S, 383R, 359A, 421T, 361H,315D, 366A, 290E, 307P and 439R, the numbering being that of the EUIndex or Kabat equivalent, and provided that mutation (i) does not takeplace on the same amino acid as mutation (ii).
 12. Polypeptide accordingto claim 1, wherein said mutated Fc region has increased affinity forthe FcgRIIb receptor (CD32b), and comprises at least one combination of2 mutations, said combination comprising: i) one mutation selected fromamong 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and ii) atleast one mutation selected from among 316D, 334R, 248E, 334N, 418P,231V, 320E, 402D, 359A, 383R, 421T and 361H, the numbering being that ofthe EU Index or Kabat equivalent, and provided that mutation (i) doesnot take place on the same amino acid as mutation (ii).
 13. Polypeptideaccording to claim 1, wherein said mutated Fc region has increased CDCactivity, and comprises at least one combination of 2 mutations, saidcombination comprising: i) one mutation selected from among 378V, 378T,396L, 421T, 334R and 326E; and ii) at least one mutation selected fromamong 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V,284L, 421T, 396L, 286I, 315D and 397M, the numbering being that of theEU Index or Kabat equivalent, and provided that mutation (i) does nottake place on the same amino acid as mutation (ii).
 14. Polypeptideaccording to claim 1, wherein said mutated Fc region has increased ADCCactivity, and comprises at least one combination of 2 mutations, saidcombination comprising: i) one mutation selected from among 378V, 326E,397M, 334N and 396L; and ii) at least one mutation selected from among316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E,394P, 307P, 378V, 378T, 286I, 286Y and 298N, the numbering being that ofthe EU Index or Kabat equivalent, and provided that mutation (i) doesnot take place on the same amino acid as mutation (ii).
 15. Polypeptideaccording to claim 1, wherein said mutated Fc region comprises at leastone combination of 3 mutations, said combination comprising: (i) onemutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M,421T, 334N, 334R, 307N and 394P; and (ii) at least 2 mutations selectedfrom among 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T,248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E,274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E,298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H,320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E,343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D,375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A,393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T,421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H,439R, 440G, 440N, 442F, 442P and 447N, the numbering being that of theEU Index or Kabat equivalent, and provided that mutation (i) does nottake place on the same amino acid as mutation (ii), preferably saidmutated Fc region comprises a combination of 4 mutations, saidcombination comprising at least one mutation (i) and at least 3mutations (ii), and preferably a combination of 5 mutations, saidcombination comprising at least one mutation (i) and at least 4mutations (ii).
 16. Polypeptide according to claim 1, wherein the parentpolypeptide comprises a parent Fc region that is a human Fc region,preferably an Fc region of a human IgG1 or human IgG2.
 17. Polypeptideaccording to claim 1, wherein the polypeptide is selected from among anisolated Fc region, a sequence derived from an isolated Fc region, anantibody and a fusion protein comprising an Fc region.
 18. Polypeptideaccording to claim 1, wherein the polypeptide consists of an Fc regionor it is an antibody.
 19. Polypeptide according to claim 1, wherein thepolypeptide is produced in the milk of transgenic animals. 20.Composition of polypeptides according to claim 1, wherein the purifiedpolypeptides of said composition, on their Asn297 glycosylation site,have N-glycans with a fucosylation rate lower than 65%, preferably lowerthan 50%, more preferably lower than 40%.
 21. Composition ofpolypeptides according to claim 20, wherein the purified polypeptides ofsaid composition, on their Asn297 glycosylation site, have a glycanstructure of biantennary type with short chains, low sialylation, havingnon-intercalary terminal mannoses and/or terminal N-acetylglucosamines.22. Composition of polypeptides according to claim 21, wherein thepurified polypeptides of said composition have a content higher than 60%for the G0+G1+G0F+G1F forms, the G0F+G1F forms being lower than 50%,preferably lower than 40%.
 23. Composition of polypeptides according toclaim 21, wherein the purified polypeptides of said composition have acontent higher than 60% for the G0+G1+G0F+G1F forms, the fucose contentbeing lower than 65%.
 24. Pharmaceutical composition comprising (i) apolypeptide according to claim 1, and (ii) at least one pharmaceuticallyacceptable excipient.
 25. A method for treating a subject in needthereof, comprising the step of administering to said subject apolypeptide according to claim
 1. 26. Antibody according to claim 18directed against an antigen selected from among a tumour antigen, viralantigen, bacterial antigen, fungal antigen, a toxin, membrane-bound orcirculating membrane, a membrane receptor.
 27. Method to produce apolypeptide comprising an Fc region and having functional activity,mediated by the Fc region, that is modified compared with that of aparent polypeptide, said method comprising a step to introduce a leastone combination of 2 mutations, said combination being selected fromamong: (i) one mutation selected from among 326E, 326T, 352L, 378V,378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least onemutation selected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L,246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M,267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T,288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G,315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R,336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R,363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S,389T, 392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R,412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K,434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, the numberingbeing that of the EU Index or Kabat equivalent, and provided thatmutation (i) does not take place on the same amino acid as mutation(ii).
 28. Method to increase the binding of a polypeptide comprising anFc region to at least one of the receptors of (FcR) of the Fc region,selected from among the receptors C1q, FcgRIIIa (CD16a), FcgRIIa (CD32a)and FcgRIIb (CD32b), said method comprising a step to introduce at leastone combination of 2 mutations, said combination being selected fromamong: (i) one mutation selected from among 326E, 326T, 352L, 378V,378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least onemutation selected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L,246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M,267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T,288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G,315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R,336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R,363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S,389T, 392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R,412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K,434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, the numberingbeing that of the EU Index or Kabat equivalent, and provided thatmutation (i) does not take place on the same amino acid as mutation(ii).